Morphologic studies have demonstrated a process by which ␣-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for ␣-granule secretion. These observations raise the possibility that morphologic features of ␣-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca ؉؉ -induced ␣-granule secretion by approximately 50%. Inhibition of ␣-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of ␣-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. IntroductionPlatelets demonstrate a number of characteristics that render them an important model of membrane trafficking. They are anucleate, display rare strands of endoplasmic reticulum, do not have Golgi structures, and synthesize little new protein. They are thought to undergo just one round of granule secretion following activation because they do not have adequate synthetic capacity to generate new granules. Although receptor-mediated 1,2 and pinocytotic 3 endocytosis have been observed in platelets, constitutive coupled endocytosis-exocytosis cycles that occur in nucleated cells in general [4][5][6][7][8] and in hematopoietic cells in particular 9 have not been documented in platelets. Thus, the degree to which generalizations regarding the molecular mechanisms of membrane trafficking can be applied to the platelet is uncertain. A second distinctive characteristic of the platelet is that its limiting membrane is characterized by a system of tunneling invaginations of the plasma membrane, termed the open canalicular system (OCS). 10,11 Evidence that the OCS is open to the extracellular environment is derived from reports using various cell-impermeant tracers. 12 The fate of the OCS on platelet activation is the subject of debate. There is evidence that the OCS undergoes evagination during platelet activation that provides an extra membrane for the formation of pseudopodia. 13,14 Others have shown th...